Review Article
The Genetic Bases of Uterine Fibroids; A Review
Veronica Medikare 1, Lakshmi Rao Kandukuri 2, Venkateshwari Ananthapur 3, Mamata Deenadayal 4, Pratibha Nallari 1*
1- Department of Genetics, Osmania University, Hyderabad, India
2- Center for Cellular and Molecular Biology, Habsiguda, Hyderabad, India
3- Institute of Genetics and Hospital for Genetic Diseases, Begumpet, Hyderabad, India 4- Infertility Institute and research Center, Secunderabad, India
Abstract
Uterine leiomyomas/fibroids are the most common pelvic tumors of the female genital tract. The initiators remaining unknown, estrogens and progesterone are considered as promoters of fibroid growth. Fibroids are monoclonal tumors showing 40-50% karyo-typically detectable chromosomal abnormalities. Cytogenetic aberrations involving chromosomes 6, 7, 12 and 14 constitute the major chromosome abnormalities seen in leiomyomata. This has led to the discovery that disruptions or dysregulations of
HMGIC and HMGIY genes contribute to the development of these tumors. Genes such as RAD51L1 act as translocation partners to HMGIC and lead to disruption of gene structure leading to the pathogenesis of uterine fibroids. The mechanism underlying
this disease is yet to be identified. The occurrence of PCOLCE amid a cluster of at
least eight Alu sequences is potentially relevant to the possible involvement of
PCOLCE in the 7q22 rearrangements that occur in many leiomyomata. PCOLCE is implicated in cell growth processes. Involvement of Alu sequences in rearrangements can lead to the disruption of this gene and, hence, loss of control for gene expression leading to uncontrolled cell growth. This can also lead to the formation of fibroids. Though, cytogenetics provides a broad perspective on uterine fibroid formation, further molecular analysis is required to understand the etiopathogenesis of uterine fibroids.
Keywords: Chromosomal translocation, Chromosomal, Estrogen, Gene rearrangement, Progesterone, Uterine fibroids, Uterine Leiomyomas (UL).
To cite this article: Medikare V, Kandukuri LR, Ananthapur V, Deenadayal M, Nallari P. The Genetic Bases of Uterine Fibroids; A Review. J Reprod Infertil. 2011;12(3):181-191.
Introduction
terine leiomyomas (UL), commonly known as fibroids, are non-cancerous tumors arising from the myometrium (smooth muscle layer) of the uterus. In addition to smooth muscle, leiomy-omas are also composed of extracellular matrix (i.e., collagen, proteoglycan, fibronectin). Leio-myomas are the most common solid pelvic tumor in women, causing symptoms in approximately 25% of women of reproductive age. However, with careful pathologic inspection of the uterus, the overall prevalence of leiomyomas increases to over 70%, because leiomyomas can be present without symptoms in many women. Leiomyomas
are usually detected in women in their 30's and 40's and shrink after menopause in the absence of post-menopausal estrogen replacement therapy (1).
Leiomyomas arise from the overgrowth of smooth muscle and connective tissue in the uterus (2). There are two components to myoma devel-opment; first the transformation of normal myo-cytes to abnormal myomyo-cytes and their growth into clinically apparent tumors. Apart from their tu-morigenic potential, they are morphologically similar at the cellular level to normal myometrial smooth-muscle cells (MSMCs). Leiomyomas may
* Corresponding Author: Pratibha Nallari,
Department of Genetics, Osmania University, Hyderabad, 500 007, India
E-mail:
prathinallari@yahoo.com
Received: Mar. 19, 2011
Accepted: Jun. 28, 2011
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have single or multiple mutated smooth-muscle tumor nodules of varying size attached and/or within the myometrium that are encircled by varying amounts of extracellular fibrous connect-ive tissue. Microscopic determinations reveal they have interlacing bundles of spindle-shaped or stellate smooth-muscle cells with little cellular pleomorphism or mitotic activity (3).
The identity of the factor(s) and molecular mechanisms involved in the cellular transforma-tion of myometrial cells into leiomyoma remains unknown. Evidence also exists supporting the involvement of genomic instability influencing genes such as estrogen and progesterone recep-tors. Several genomic and proteomic studies have also provided evidence for altered molecular environment of leiomyomas compared to the nor-mal myometrium, as a possible biomarker in their proliferation and regression (4).
Global gene expression profiling of uterine leiomyomas (ULMs) revealed that hundreds of genes were dysregulated including those with functional roles in cell proliferation, differenti-ation and extracellular matrix production. So far, only a few specific genes or cytogenetic aberra-tions have been identified to be associated with ULMs. While many of the dysregulated genes may function as either effectors or promoters of ULMs growth, they are likely to be secondarily induced and indirectly responsible for tumor growth into morbid and symptomatic ULMs (5).
Classification of leiomyomas: Leiomyomas are classified by their location in the uterus. Sub-serosal leiomyomas are located just under the uterine serosa and may be pedunculated (attached to the corpus by a narrow stalk) or sessile (broad-based). Intramural leiomyomas are found predom-inantly within the thickness of myometrium but may distort the uterine cavity or cause an irregular external uterine contour. Submucous leiomyomas are located just under the uterine mucosa (endo-metrium) and, like subserosal leiomyomas, may be either pedunculated or sessile. Tumors in sub-serosal and intramural locations comprise the majority (95%) of all leiomyomas; submucous leiomyomas make up the remaining 5% (1).
The clinical sequelae of leiomyomas, which may depend on their location within the uterus, may be associated with a spectrum of symptoms, includ-ing excessive menorrhagia, severe abdominal
pain, urinary incontinence, and constipation. Leio-myomas may lead to infertility, spontaneous abor-tions, premature labor, or dystocia. Progression of leiomyoma to malignant leiomyosarcoma (LMS) is very rare (6).
The present review focusses on the cytogenetic and molecular aspects which contribute to the pathogenesis of uterine leiomyomas. Here the genetics of leiomyomata and the current under-standing of cytogenetic abberations and gene ex-pression with respect to their contributions to the development of these tumors is summarized.
Risk factors: Risk factors are characteristics associated with a condition generally identified by epidemiological studies. There is a suggestion of slightly increased risk of fibroids associated with early menarche (7 - 9). The early onset of men-strual cycles may increase the number of cell divisions that the myometrium undergoes during the reproductive years, resulting in an increased risk for mutations in gene/s controlling myome-trial proliferation (10, 11).
An increase in age has been demonstrated to increase the prevalence of fibroids during the reproductive years (12). Hormonal factors associ-ated with pre-menopause may be important modulators for the development of fibroids during late reproductive years; alternatively this may also be due to the cumulative culmination of 20-30 years of stimulation by estrogen and progesterone. The other factors involved are menopause, obes-ity, diet, exercise, racial differences, geographic differences, oral contraceptives, hormone replace-ment theory, etc (13).
Apart from these, hyperinsulinemia is con-sidered as a risk factor since insulin may influence UL development through direct promotion of myometrial smooth muscle cell proliferation or by increasing circulating levels of ovarian hormones (14).
Theories of fibroid formation: Despite the major public health impact of leiomyomas, little is known about their cause. The most important aspect of the etiology of fibroids - the initiator(s)-remains unknown. Several theories have been advanced. One hypothesis states that increased levels of estrogen and progesterone result in an increased mitotic rate that may contribute to myoma formation by increasing the likelihood of somatic mutations (15). Another favors an
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ent abnormality in the myometrium of individuals who develop fibroids, based upon the finding of significantly increased levels of estrogen receptors (ER) in the myometrium of fibroid uteri (13).
More recently, growth factors have been shown to mediate the growth-promoting effects of estro-gen and play an important role in the development of fibroid tumors (1). Growth factors, proteins/ polypeptides produced locally by smooth muscle cells and fibroblasts control the proliferation of cells and appear to stimulate myoma growth, primarily by increasing the extracellular matrix. Some of the identified myoma-related growth
factors are transforming growth factor-β (TGF-β),
basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), and prolactin (16).
Growth factors affect cells in complex ways, and the response to combinations of growth fac-tors may be different from the response to an individual factor. Many of these growth factors are overexpressed in myomas and either increase
smooth muscle proliferation (TGFβ, bFGF),
in-crease DNA synthesis (EGF, PDGF), stimulate
synthesis of extracellular matrix (TGF-β),
pro-mote mitogenesis (TGF-β, EGF, IGF, prolactin),
or promote angiogenesis (bFGF, VEGF) (16). The steroid hormones, estrogen and progester-one, are considered the most important regulators of leiomyoma growth.
Estrogen: There is abundant evidence that estro-gen promotes fibroid growth, including the clinic-al observations that fibroids grow in the presence of high levels of estrogen, such as during the reproductive years, or they regress in the presence of low levels of estrogen, such as following menopause or during gonadotropin releasing hor-mone (GnRH) agonist therapy (1).
Furthermore, because the risk of fibroids is greater in nulliparous women who might be sub-ject to a higher frequency of anovulatory cycles and obese women with greater aromatization of androgens to estrone in the adipose tissue, the concept of unopposed estrogens, as an underlying cause of uterine fibroids, has been proposed in the literature (17, 18). Increased growth of myomas among women taking tamoxifen or receiving transdermal or injectable estrogen replacement
therapy further supports the importance of estro-gen. The estrogen hypothesis has also been sup-ported by clinical trials evaluating the medical treatment of myomas with GnRH agonists, the effective result of which is hypoestrogenism accompanied by regression of the fibroids (19).
Progesterone: Progesterone is also thought to play a role in fibroid growth which is supported by clinical studies. For example, fibroid size increases during treatment with synthetic proges-terones. In contrast to GnRH agonist therapy alone which has been shown to reduce uterine volume, combination of GnRH agonist and pro-gesterone therapy has been shown to have no effect on uterine volume. The observation that fibroids regress with the administration of anti-progesterone agent, RU-486, further supports the role of progesterone as a promoter of fibroid growth. Histologically, fibroids from patients treated with progesterone show more cellular growth than those from patients without proges-terone therapy. Altogether, these data suggest that progesterone also enhances fibroid growth (1).
Estrogen and progesterone receptors: In the major-ity of the reviewed studies, the concentrations of both the estrogen receptors (ERs) (at 6q25.1 and 14q) and progesterone receptors (PRs) (at 11q22) were greater in leiomyomata than the myometri-um. In addition, researchers found the ER and PR to be elevated in fibroids during all phases of the menstrual cycle when compared with matched myometria. Interestingly, in one study the ER and PR levels were significantly higher in submucous than subserosal leiomyomas, leading the authors to speculate about different etiologies and types of leiomyomas (13).
Leiomyomas as clonal tumors: X-inactivation studies: Analyses of multiple leiomyomas from a single uterus have demonstrated that the tumors can harbor different chromosomal changes and suggest that each tumor can develop independent-ly. X-inactivation studies, based on the phenom-enon of lyonization, i.e., inactivation of one X-chromosome in normal female cells, have demon- strated that leiomyomas develop as clonal lesions. Initially, glucose-6-phosphate dehydrogenase (G6 PD) isoenzyme analysis was used to demonstrate the independent clonal origin of multiple tumors in a single uterus (6).
Another, more informative, approach based on
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the CAG repeat polymorphism in the X-linked androgen receptor gene has been used to examine clonality and the results confirmed the mono-clonal nature of leiomyoma. A study of a patient with 2 independent leiomyomas, each showing a different pattern of X-chromosome inactivation but with identical del(7)(q21.2q31.2) derivative chromosomes, supports the view that identical cytogenetic changes in multiple leiomyomas from the same patient may represent recurrent chromo-somal aberrations in smooth muscles or they may be coincidental. Cytogenetically mosaic tumors were also reported to be clonal (6).
Studies on the inactivation of X-chromosome-linked phosphoglycerokinase (PGK) showed that all studied leiomyomas had a single type of inactive allele and were of unicellular origin but independently generated in the uterus (6). Based on the monoclonal pattern of X- inactivation in the karyotypically normal cells in leiomyomas,
Mashal et al. (20)suggested that these cells were
part of the tumor clone and that clonal expansion of tumor cells might precede the development of cytogenetic changes in some leiomyomas.
The discovery of heterogeneity of chromosomal aberrations is consistent with the multistep hy-pothesis of tumor development, in which the function (or dysfunction) of several genes at multiple loci results in fibroid growth. Abnormal-ities at several loci have been documented in individual tumors, and this heterogeneity may explain clinicopathologic differences seen in fibroids, including variation in size or response to hormonal treatments. (21).
Cytogenetic studies: Standard karyotyping has been used to detect chromosomal aberrations such as deletions, duplications and translocations which require the culture of leiomyoma cells to obtain metaphase preparations.
An alternative method that has been employed in a few studies (22, 23) is comparative genome hybridization, which permits the recognition of cytogenetic changes such as deletions and amp-lifications without the need for cell cultures, although it does not allow detection of balanced rearrangements. Neither standard karyotyping nor comparative genomic hybridization permits the detection of small submicroscopic chromosomal abnormalities such as point mutations or epigenet-ic changes such as methylations (13).
Approximately 40% of UL have non-random and tumor-specific chromosome abnormalities. This has allowed classification of some UL into well-defined subgroups which include deletion of portions of 7q, trisomy 12 or rearrangements of 12q15, 6p21 or 10q22. Additional abnormalities, which appear consistently but not as frequently, include rearrangements of chromosomes X, 1, 3 and 13. The variety of chromosomal rearrange-ments, including but not limited to translocation, deletion and trisomy, predict different molecular genetic mechanisms for UL formation and growth (24). A tendency for karyotypically abnormal lei-omyomas to be more cellular and to have a higher mitotic index than do chromosomally normal tumors, has been reported (6).
Although no relationship between the patient age or parity and the type of chromosomal abnor-mality has been identified, a few studies have found a positive correlation between the presence of a cytogenetic abnormality and the anatomic location of uterine leiomyomas, i.e., intramural (35%) and subserous (29%) leiomyomas are more likely to have abnormal karyotypes than the sub-mucous (12%) type. Another study, showed a relationship between karyotype and leiomyoma size, with the largest tumors carrying t(12;14) abnormalities. In contrast, tumors with del(7) were found to be smaller and those with mosaic karyotypes were intermediate in size (13).
The abnormal karyotypes in leiomyoma were frequently accompanied by 46, XX, i.e., cytogen-etically normal female cells. Although this may represent concomitant growth of normal cells, it is possible that these apparently normal cells may have undergone neoplastic transformation, with the chromosome changes appearing subsequent to this transformation (6).
The t(12;14)(q15;q23~q24) in leiomyoma: The most common chromosomal aberration in leiomy-oma, seen in approximately 20% of karyotypically abnormal leiomyomas, is the characteristic trans-location, t(12;14) (q15;q24), specifically associ-ated with leiomyoma. Other rearrangements in- volving 12q14~q15, such as paracentric inver-sions, have also been reported. Rearrangements involving the same region of 12q in leiomyoma and other benign solid tumors (e.g., angiomyxo-mas, breast fibroadenoangiomyxo-mas, endometrial polyps, hemangiopericytomas, lipomas, pulmonary
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droid hamartomas, and salivary gland adenomas) support the notion that critical genes reside within 12q14~q15 (6).
Besides involvement of chromosome 12 in the t(12;14), trisomy 12 is not an uncommon cytog-enetic change in leiomyomas. Leiomyomas with normal karyotypes may have cryptic inversions of 12q. Though 12q15 is often part of t(12;14) in leiomyoma and a der(14)t(12;14)(q15;q23~q24) is seen in most leiomyomas, rearrangements of chromosomes 1, 5, 8, and 10 do not infrequently accompany those of 12q15 (6).
Chromosome 7 changes in leiomyoma: Several karyotyping studies have identified the region 7q22–q32, comprising about 30 Mbp, as being commonly deleted (25). Early studies reported deletions of chromosome 7 from band q11.23 to q36. (21). Although interstitial deletions and translocations involving chromosome 7q have also been reported in lipomas and endometrial polyps, the deletion is more commonly observed in fibroids than in any other solid tumor (13). Previous reports of inversions and translocations involving 7q22 suggest that a region within this cytogenetic band is critical for the pathobiology of uterine leiomyomas (21). In addition, del(7) may be associated with t(12;14) or t(1;6), suggesting involvement of del(7) in the karyotypic evolution of leiomyoma (6).
An interstitial deletion of chromosome 7, del(7) (q22q32) is observed with a frequency of about 17% in karyotypically abnormal leiomyomas. Lei-omyomas with chromosome 7 deletions or trans-locations are usually found in the mosaic state with 46, XX cells. Apparent negative selection for these aberrant cells in tissue culture results in frequent loss of the del(7)(q22q32) cells. Cells with the del(7)(q22q32) are more likely to persist in cultures when the t(12;14) is also present (6).
The instability of del(7)(q22q32) cells without chromosome 12 aberrations is intriguing in light of the observation that fibroids with chromosome 12 abnormalities or rearrangements are often larger than tumors with chromosome 7 abnormal-ities, and larger fibroids are more likely to be chromosomally abnormal than smaller ones. Observations of tumor instability in culture and decreased tumor size in association with the del(7) (q22q32) suggest that a gene regulating cellular growth may reside in 7q22. Dissection of 7q22 to
study leiomyoma-specific sequences is compli-cated by the fact that this is a gene-dense region including genes involved in developmental
pro-cesses (DLX5, DLX6) and collagen metabolism
(collagen type 1, procollagen C-endopeptidase enhancer), as well as those that encode acetyl-cholinesterase, plasminogen activator inhibitor type 1, and mucin (21).
The occurrence of deletions in both chromo-some homologues in the same region strongly suggests the involvement of a tumor suppressor gene in the development or progression of uterine leiomyomas (26).
Rearrangements of 6p21 in leiomyoma: Rearrange-ments of 6p21 have been observed in benign mesenchymal tumors, including lipomas, pulmon-ary chondroid hamartomas, endometrial polyps, and leiomyomas. In leiomyomas, these rearrange-ments occur with a frequency of 5% and include t(1;6)(q23;p21), t(6;14)(p21;q24), and t(6;10) (p21;q22), as well as inversions and translocations with other chromosomes (6).
Other cytogenetic abnormalities: Other cytog-enetic abnormalities of lower frequency than those above have been reported in leiomyomas. These include changes of the X-chromosome, including del(X)(p11.2), t(X;12)(p22.3;q15), -X, der(5)t(X;5)(p11;p15), del(X)(q12), der(X)t(X;3) (p22.3;q11.2), and inv(X)(p22q13). Although ei-ther the short or long arm of the X chromosome is rearranged, the region Xp11~p22 may be prefer-entially involved. Structural rearrangements of chromosome 1 in leiomyoma, particularly in the form of ring chromosomes usually occur con-comitantly with other chromosomal changes and are, therefore, thought to represent secondary abnormalities. Other structural aberrations of chromosome 1 detected in leiomyoma include t(1;6)(q23;p21) and t(1;2)(p36;p24). Monosomy 10 and deletions of 10q (especially band q22) have been detected in leiomyoma. A number of rearrangements of chromosome 3 have been observed in leiomyoma, both as sole abnormal-ities or accompanying other rearrangements in-cluding: ins(2;3)(q31;p12p25), del(3)(p14), del(3) (q24), and t(3;7)(p11;p11). A subgroup of leiomy-omas demonstrating aberrations of 13q has been described with deletions in that arm possibly playing a primary role in the development of some leiomyomas (6).
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Molecular studies in uterine leiomyoma: In studies on the molecular genetic changes in leiomyomas, much attention has been given to the possible involvement of genes located in chromosomal regions affected by recurrent changes, which are discussed below.
HMGA genes: High-mobility group I (HMGI)
proteins, including those coded by the HMGA2
(HMGIC) and HMGA1 (HMGIY) genes, are
expressed predominantly during embryonic devel-opment and are generally silenced in adult tissues. The HMGA2 and HMGA1 genes encode closely related, low-molecular-mass proteins; HMGA proteins which are capable of binding to the minor groove of AT rich DNA with three DNA-binding domains (the so called AT-hooks), thus inducing conformational changes in chromatin structure and enabling the regulation of the expression of various target genes. In addition, they can interact with other proteins by means of their acidic domain (27).
HMGA1 and HMGA2 map to chromosomal
bands that are targeted by non-random structural chromosomal abnormalities found in uterine
leio-myomas, i.e., 6p21 for HMGA1 (28) and
12q14-15 for HMGA2 (29, 30). Usually, these regions
are affected by chromosomal translocations but inversions can occur as well with structural chromosomal aberrations affecting 12q14-15, being much more frequent than those affecting 6p21. The molecular alterations resulting from the cytogenetic deviations generally seem to include an upregulation of the genes (31, 32).
HMGA2 and HMGA1 are both closely related as well as highly conserved (96% conservation
between mouse and human HMGA2 homologues)
(21).
HMGA2: The entire HMGA2 gene spans
ap-proximately 160 kb, including a 327 bp coding
region, an 854 bp 5' untranslated region (UTR)
and a 2966 bp 3' UTR, which together encode a
4.1 kb mRNA. The first three exons of HMGA2
are clustered within 13 kb of each other and
together they encode a DNA-binding domain comprising the AT hook motif. A large intron
(approximately 140 kb) between exons 3 and 4
separates the DNA-binding domains from the rest of the protein. Exon 4 encodes an 11-amino acid
spacer domain that is absent in HMGA1 and
precedes a 13 kb intron that separates it from the
acidic domain encoded by exon 5 (21).
Chromosomal breakpoints have been assigned to regions outside the open reading frame of
HMGA2 gene, thus primarily affecting its
expression rather than its protein sequence (33).
Previous studies have shown that HMGA2 is
abnormally overexpressed in many benign and malignant neoplasms, particularly in uterine leiomyomas. Although its role in tumorigenesis is
not well understood, overexpression of HMGA2 is
found to be associated with large tumor size in leiomyomas, suggesting its role in promoting tumor growth (34). Some leiomyomas
overex-press the HMGA2 structural transcription factor,
which has been shown to cause dysregulation of several genes in leiomyomas. However, no correl-ations have been found between the levels of
EGR-1 and HMGA2 in leiomyomas. Furthermore,
HMGA2 transcripts may be truncated in the
COOH-terminal in leiomyomas due to chromo-some 12 changes and still be pathogenetic. A
comparison of HMGA2 expression in leiomyomas
versus LMS of various origins revealed a higher percentage of the former (70% vs. 58%). Relevant observation is trisomy 12 in leiomyomas, which
would potentially upregulate HMGA2 expression
by a gene dosage mechanism (6).
MicroRNAs such as let-7 family are important small molecules known to participate in the
fine-tuning of functional gene activity (34). HMGA2 is
also predicted as one of the genes targeted by the let-7 family, and overexpression of let-7b in leiomyoma cells resulted in the suppression of
HMGA2 expression (35). HMGA2 is one of the major targets of Let-7 microRNAs, and large
leiomyomas have high levels of HMGA2, hence, it
is speculated that dysregulation of the Let-7:HMGA2 regulatory mechanism may be one of key genetic events promoting leiomyoma growth.
Characterization of Let-7 repression of HMGA2 in
leiomyomas can provide a better understanding of
HMGA2 function. HMGA2 overexpression and a loss of Let-7 microRNA expression are more common in large leiomyomas. Peng et al. proposed that disrupting the pairing between
Let-7 and HMGA2 was an important molecular
mechanism in promoting leiomyoma growth (34).
HMGA1: The smaller (10 kb) HMGA1 gene con-
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sists of eight exons: exons 1 to 4 are noncoding, exons 5 to 7 contain the AT hooks corresponding
to exons 1 to 3 of HMGA2, and exon 8 encodes
the acidic C-terminus of the protein. Alternate splicing of this gene results in two products, one
of which is a variant lacking a 33 bp segment
between exons 5 and 6, which is also absent from
HMGA2. Despite the great structural similarity
between HMGA2 and HMGA1, expression
pat-terns of the two proteins are strikingly dissimilar, perhaps suggesting the existence of distinct regulatory elements as well as different functional roles (21).
The HMGA1 gene has been mapped to 6p21, a site of recurrent chromosomal rearrangements in benign mesenchymal tumors, e.g. lipoma, hamar-toma, and leiomyoma. HMGA1 protein is known
to bind to specific AT-rich domains and promoters
of a number of diverse genes. A single genomic
clone including HMGIY was found to span the
breakpoint in a uterine leiomyoma with a complex rearrangement involving chromosome region 6p21.3 (36). Subsequent studies demonstrated
rearrangement of the HMGIY locus in hamartoma
of the breast (37), a pericentric inversion of chromosome 6 involving band p21 in a uterine leiomyoma (38), and a translocation leading to an
intergenic fusion with the LAMA4 gene (laminin
a4) in a pulmonary chondroid hamartoma (39).
RAD 51L1: RAD51L1, a member of recombin-ation family mapping to 14q23-24, is a strong
candidate as a translocation partner for HMGA2 in
leiomyoma. RAD51L1/HMGIC chimeric
tran-scripts were first identified in SV-40 transformed cell lines containing this translocation (40).
Ingraham et al. (41) found the premature
trunca-tion of the RAD51L1 major transcript in the
primary culture of a leiomyoma. The RAD51L1 protein is physically associated with other RAD51-like proteins and contains motifs for nucleotide binding, which may play a role in cell
cycle regulation and apoptosis. RAD51L1 plays a
role in DNA repair recombination. RAD51L1
phosphorylates a number of proteins, including TP53, cyclin E, and CDK2, suggesting that
RAD51L1 affects cell cycle progression (6).
Ingraham et al 1999 speculated that disruption
of the RAD51L1 gene involved in the t(12;14)
(q15;q24), results in the loss of the exon encoding
the 3′ end of the predominant exon of that gene
which might be an important step in the early pathogenesis of leiomyoma. They were unable to detect expression of a fusion gene resulting from the t(12;14) and speculated that alterations of the relative expression levels of the various
RAD51L1 isoforms may be a significant effect of
the translocation. Although, studies showed that RAD51L1 is directly interrupted by a transloca-tion breakpoint in only one case, translocatransloca-tion breakpoints previously characterized in 2 other leiomyomas map within the BCR that contains this gene, and could affect its expression through alteration of the translocated chromosome (41).
The majority of 12q15 breakpoints in
leiomy-omas were found to be located 5′ in the HMGA2
locus, a minority at the 3′ region (usually outside
the HMGA2 coding region) or with 5′ and 3′
breakpoints flanking HMGA2. Fusion transcripts
of 5′ region of HMGA2 with the 3′ region of
RAD51L1 were detected with a breakpoint 3′ to
HMGA2; the derived sequences indicated that the
breakpoints resided within the 3′ untranslated
re-gion of HMGA2 with the fusion transcripts
con-taining the full coding sequence of HMGA2 (6).
Though fusion transcripts involving HMGA2
and RAD51L1 were seen in a small percentage of
leiomyomas with 5′ breakpoints, HMGA2
trans-cripts as well as those of RAD51L1 were present
in all tumors with t(12;14). With the breakpoint 5′
(centromeric) relative to HMGA2, the entire gene
should be translocated to the der (14), and all of
the sequence telomeric to HMGA2 should be
derived from chromosome 12. Exons 8–11 of
RAD51L1 included in a potential HMGA2/
RAD51L1 fusion product would be located either
upstream (i.e., centromeric) to HMGA2 on the
der(14) or on the der(12) remote from HMGA2,
depending on the position of the chromosome 14 breakpoint (6).
Partner genes for HMGA2 other than RAD51L1
are: the COX6C gene at 8q22~q23, the ALDH2
gene at 12q24.1, the enhancer of invasion (HEI10)
gene at 14q11, and the RTVL-H 3′ gene on
chromosome 12, respectively. It is possible that these leiomyomas were associated with either variant translocations involving chromosome 12 with a chromosome other than 14, or with a complex translocation with a chromosome add-itional to chromosomes 12 and 14 or a chromo-somal band other than 12q14~q15 (6).
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PCOLCE: The human PCPE gene (PCOLCE) has been mapped to 7q22 (42), an area frequently disrupted in uterine leiomyomata (43). It is a
small gene measuring 6.0 kb with a conserved
intron/exon structure comprising 9 exons. Where-in the NH2-termWhere-inal CUB domaWhere-in (CUB1) is encoded by exons 2 and 3, whereas the second CUB domain (CUB2) is encoded by exons 4 and 5 and by part of exon 6. The remainder of exon 6 encodes a region that shows low sequence
hom-ology between human and mouse PCPEs. This
region probably represents a “linker” region that connects the two conserved and functionally distinct portions of PCPE: the NH2-terminal CUB domains that provide full C-proteinase enhancing activity and the COOH-terminal domain that appears capable of inhibiting matrix metallopro-teases.3. The COOH-terminal domain is encoded
by the last three exons of PCOLCE. In PCOLCE
the 3′ exon contains 164 bp of coding sequence
and 71 bp of 3′ untranslated region (42).
The occurrence of PCOLCE amid a cluster of at
least eight Alu sequences is potentially relevant to
the possible involvement of PCOLCE in the 7q22
rearrangements that occur in many leiomyomata, as recombination events involving Alu sequences have previously been found to be involved in generating various gross rearrangements of genes
and chromosomes (44). The PCPE gene has been
implicated in the control of cell growth and differentiation, and the chromosomal location of
PCOLCE at 7q22 places it at a site where chromo-somal rearrangements are associated with leiomy-omata (43).
Transforming growth factor-β: The TGF-β family comprises five dimeric polypeptides encoded by
distinct but closely related genes. TGF-βs are
multifunctional growth factors that regulate many aspects of cellular function including prolifer-ation, differentiprolifer-ation, ECM production, and chemotaxis (45). Expression of all the three types
of TGF-β, as well as TGF-β receptors I–III, has
been detected in human myometrial tissue (13).
TGF-β3 has been shown to cause increased
proliferation of both leiomyoma and myometrial cells, and adherence of a neutralizing antibody to this peptide causes a decrease in proliferation. Leiomyomas express up to six fold higher levels
of mRNA for TGF-β3 than do normal myometrial
cells at all stages of the menstrual cycle (45). The
expression of transforming growth factor β
(TGF-β) receptor type II is the target of miR-21 in
leiomyoma smooth muscle cells (LSMCs). TGF-β
is a key profibrotic cytokine that mediates its
biological activities by binding to TGF-β receptor
(TGF-βR) types I to III, of which types I and II
are transmembrane proteins with a cytoplasmic
serine/threonine kinase domain. TGF-β and
TGF-β receptors as well as their intracellular signaling
pathways are overexpressed in leiomyoma com-pared with that in myometrium. The consequence of a lower expression of miR-21 in leiomyoma and LSMCs compared with that in myometrium might represent the loss of one of the regulatory mechanisms resulting in unregulated expression
of TGF-β receptor and increased TGF-β activities
(4).
Thus, TGF-β3 may contribute to the enhanced
growth of leiomyomas by increasing cell prolifer-ation as well as potentially increasing ECM production (45). In view of the probable role of
this growth factor in fibroid pathophysiology, it is
of particular interest that the gene coding for
TGF-β3 is located near the 14q23-24 breakpoints,
one of the most common translocation sites
identified in cytogenetic studies of fibroids (13).
Conclusion
Uterine fibroids are the most common benign
tumors of the female genital tract, with the etiology of leiomyomas still remaining unknown. Cytogenetic abnormalities indicate different gen-etic pathways by which fibroids grow and de-velop. Although most of uterine leiomyomas have a normal karyotype, reports suggest that 50% of
these tumors bear specific chromosomal
aberra-tions that include chromosome 3, 6, 7, 13, trisomy 12, reciprocal translocation between chromo-somes 12 and 14 and monosomy 22. Such chromosomal rearrangements may be responsible for initiation as well as the growth of these tumors.
The cytogenetic abnormalities indicated to date shows genetic heterogeneity among these tumors. Further molecular analyses would help to identify putative candidate genes in uterine leiomyomata formation. Identification of genes involved may aid in the genetic diagnosis of the condition, prediction of genetic risks and management of the condition by appropriate therapeutic measures.
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Alu elements have been associated with genetic diseases, mainly by providing sites for homolo-gous recombination which result in deletions or other more complex genomic rearrangements. This phenomenon can lead to evolution of novel karyotypes. Since Alu sequence influences rear-rangement, the candidate genes disrupted by these rearrangements in UL can be mapped using BAC clones in the form of cytochips. With the advent of microarray technology, the development of cytochips can help to detect abnormalities with a much greater resolution. In addition to identifying the genomic changes, the use of cytochips would give a better understanding of genes which are involved in the pathogenesis of UL. This informa-tion would help in identifying genotype-pheno-type correlations.
An important avenue of research involves
studying affected women and their first-degree
relatives who also have uterine fibroids. The
availability and examination of such individuals not only will hasten cytogenetic and molecular studies, but will also be crucial to dissecting and
defining the genetic loci that undoubtedly
contrib-ute to the development of contrib-uterine leiomyomata. The development of uterine fibroids involves complex interaction among genes and environ-ment; further understanding and uncovering of the genetic and environmental mechanisms respon-sible for uterine leiomyoma will aid in designing rational gene-therapy protocols.
On the basis of our current state of knowledge, one can only speculate upon the initiators of this common condition. Future research efforts may provide a better understanding of the causes and mechanisms of uterine fibroid tumorigenesis. Insights resulting from elucidation of the basic biology of these tumors might then be successful-ly translated into preventative strategies that will reduce the incidence and/or morbidity of this disease.
Acknowledgement
Acquisition of Financial support from the Uni-versity Grants Commission, New Delhi is ac-knowledged. Authors declare no conflict of inter-est.
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